Coated water soluble nanoparticles

ABSTRACT

Nanoparticles and methods of making nanoparticles are provided. The nanoparticles may include semiconductor nanocrystals. A shell may encapsulate a nanoparticle core, and the shell may include non-organic material and may be silica. The shell may also include additional species such as PEG. In some embodiments, a passivation layer is in contact with the core.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/144,070, entitled “Method of Forming Coated Water-SolubleNanoparticles by Inverse Emulsion” and having a filing date of Jun. 23,2008, which is a divisional of U.S. patent application Ser. No.10/911,402, entitled “Coated Water Soluble Nanoparticles” and having afiling date of Aug. 4, 2004, which are incorporated herein by referencein their entirety for all purposes.

BACKGROUND OF INVENTION

1. Field of the Invention

The invention relates to nanoparticles and methods for makingnanoparticles, and in particular, to semiconductor nanocrystals thatexhibit improved hydrophilicity.

2. Discussion of Related Art

Nanoparticles are microscopic particles of matter having dimensions onthe nanometer scale. Of particular interest are a class of nanoparticlesknown as semiconductor nanocrystals, or quantum dots, that exhibitproperties that make them particularly useful in a variety ofapplications. Because of quantum confinement effects, semiconductornanocrystals can exhibit size-dependent optical properties. Theparticles give rise to a class of materials whose properties includethose of both molecular and bulk forms of matter. When thesenanoparticles are irradiated, more energy is required to promote theelectrons to a higher state, leading to an increase in energy release inthe form of photons and light emission in a color that is characteristicof the material. The resulting photons that are released typicallyexhibit a shorter wavelength than those released from a bulk form of thesame material. The quantum confinement of electrons and holes in threedimensions contributes to an increasing effective band gap withdecreasing nanocrystal size. Therefore, smaller nanocrystals typicallyexhibit shorter emitted photon wavelength. For example, nanocrystals ofcadmium selenide (CdSe) can emit across the entire visible spectrum whenthe size of the crystal is varied over the range of from two to sixnanometers.

Another aspect of semiconductor nanocrystals is that crystals of auniform size typically are capable of a narrow and symmetric emissionspectrum regardless of excitation wavelength. Thus, if nanocrystals ofdifferent sizes are employed, different emission colors may besimultaneously obtained from a common excitation source. Thesecapabilities contribute to the nanocrystals' potential as diagnostictools, for example, as fluorescent probes in biological labeling anddiagnostics. These nanocrystals, or quantum dots, exhibit high emissionstability over long periods of time, thus providing advantages overconventional biological probing dyes. One class of semiconductornanocrystals are the cadmium chalcogenides. These include, for example,cadmium selenide and cadmium telluride nanoparticles.

It is known that improved quantum yields of semiconductor nanocrystalscan be obtained by passivating the nanocrystals by reducing the incidentof surface non-radiative recombination sites. Surface passivation can beachieved, for example, by coating a material around the nanocrystals.See, e.g., Alivisatos et al., U.S. Pat. No. 6,255,198. The coatings canbe inorganic or organic although inorganically coated quantum dots aretypically more robust and exhibit less degradation of photo luminescencequantum yield in solution than do organically passivated quantum dots.

For semiconductor nanocrystals to be useful in biological applications,it is preferred that the crystals are water soluble, photo-stable andnon-toxic. Some quantum dots may exhibit water solubility but aretypically not photo-stable and are toxic. Other nanocrystals have beencoated, for example, with short chain water soluble molecules, such asthiols, to render the nanocrystals soluble. However, these organicallycoated quantum dots have been shown to be unstable and exhibitdeteriorating photo-luminescent properties. Others, such as Bawendi etal. in U.S. Pat. Nos. 6,319,426 and 6,444,143, hereby incorporated byreference, have synthesized semiconductive nanocrystals having anorganic layer that also includes linking groups for the attachment ofhydrophilic groups that can provide improved water solubility.

Some have proposed coating nanocrystals using silicate as a precursor.These methods use silane as a surface primer to deposit a thin shell ofsilica in water. The silica shell can then be thickened using the Stöbermethod. These procedures, however, are complicated and time-consuming.Others have used microemulsions as a technique for silica coating. Inparticular, using reverse microemulsions, monodispersed silica particlescan be synthesized. Encapsulation of nanoparticles within silica canlead to an enhancement in chemical stability and photo-stability. Thishas been done in nanoparticles having a zinc sulfide (ZnS) core/twophoton dye/silica particles and the encapsulated dye within the silicashell has exhibited enhanced luminescence and lifetime. However,synthesized TOPO semiconductor nanocrystals are water insoluble and thussilica cannot be precipitated with the nanocrystals within the aqueousdomains of the microemulsion.

SUMMARY OF INVENTION

The invention is directed, in part, to nanoparticles, solublenanoparticles and methods for making nanoparticles.

In one aspect, a coated nanoparticle is provided, the nanoparticlecomprising a core comprising a semiconductor material, anon-semi-conductor passivation layer contacting at least a portion ofthe core, and a non-organic shell encapsulating at least partially thecore and the passivation layer.

In another aspect, a method of making a water-soluble nanoparticle isprovided, the method comprising contacting an amine with a nanoparticleto modify the nanoparticle surface, suspending the nanoparticle in anaqueous-in-nonaqueous emulsion, introducing a silica precursor to theemulsion, and polymerizing the silica precursor to form a silica shellthat at least partially encapsulates the nanoparticle.

In another aspect, a semiconductor nanocrystal solution is provided, thesolution comprising an aqueous solution having a pH of less than about8.0, a plurality of semiconductor nanocrystals dissolved in the aqueoussolution wherein at least 90% of the semiconductor nanocrystals, byweight, remain dissolved for greater than 6 hours.

In another aspect, a semiconductor nanocrystal is provided wherein thenanocrystal is soluble in water at a pH of less than about 8.0.

In another aspect, a nanoparticle is provided, the nanoparticlecomprising a silica shell encapsulating a core, the silica shellincluding polyethylene glycol, or a derivative thereof.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a schematic illustration of a silica-coated CdSe semiconductornanocrystal.

FIG. 2 is a schematic illustration of a coated nanocrystal with a shellincluding long chain hydrophilic species.

FIG. 3 is a schematic illustration of a nanoparticle including a shelland a passivation layer.

FIG. 4 illustrates a nanoparticle core in contact with an amino silanesurface cap and a surfactant.

FIG. 5 illustrates a nanoparticle core with exchanging surfactants and asilica shell.

FIGS. 6 a-6 c provide a schematic diagram describing the encapsulationof hydrophobic semiconductor nanocrystals within the aqueous domains ofa reverse microemulsion, using the interaction of hydrophilic groups(polar ends) of two different surfactants, TOPO and IGEPAL.

FIGS. 7 a-7 c provide a schematic diagram describing steps that canresult in the encapsulation of hydrophobic semiconductor nanocrystals(QDs) within a silica shell. The steps include micellar collisions,nucleation and polymeric growth of monomers.

FIG. 8 is a photocopy of a transmission electron micrograph (TEM) ofCdSe/ZnS QD-silica core-shell structures having a diameter of about 6 nmencapsulated within an approximately 22 nm silica shell using amicroemulsion technique. The majority of single QDs are encapsulatedwithin a single silica shell.

FIG. 9 is a photocopy of a transmission electron micrograph (TEM) ofCdSe/ZnS QD-silica core-shell structures having a diameter of about 6 nmencapsulated within an approximately 100 nm silica in the microemulsion.

FIG. 10 illustrates graphically the effect of TEOS concentration onsilica shell thickness and also on the quantum yield (QY) relative touncoated CdSe/ZnS nanoparticles.

FIG. 11 shows the absorption and emission spectra of CdSe/ZnSsemiconductor nanoparticles with and without silica coating.

FIG. 12 graphically compares the photo-stability of silica-coatedCdSe/ZnS semiconductor nanocrystals with organic-coated semiconductornanoparticles.

FIGS. 13 a-c illustrate the changes of emission intensity of coresemiconductor nanocrystals with various silica coating times. Volume ofTEOS (V_(TEOS)) added into the microemulsion is indicated.

FIG. 14 a shows the relative emission intensity versus coating time atvarious TEOS concentrations for core semiconductor nanocrystals.

FIG. 14 b shows the emission wavelength versus coating time at variousTEOS concentrations for core semiconductor nanocrystals.

FIG. 14 c shows the relative emission intensity versus TEOSconcentrations at 2 hours coating time for core semiconductornanocrystals.

FIGS. 15 a-c illustrate the changes of emission intensity ofAPS-modified core semiconductor nanocrystals upon various silica coatingtimes. Volume of TEOS (V_(TEOS)) added into the microemulsion isindicated.

FIG. 16 a shows the relative emission intensity versus coating time atvarious TEOS concentrations for APS-modified core semiconductornanocrystals.

FIG. 16 b shows the emission wavelength versus coating time at variousTEOS concentrations for APS-modified core semiconductor nanocrystals.

FIG. 16 c shows the relative emission intensity versus TEOSconcentrations at 8 hours coating time for APS-modified coresemiconductor nanocrystals.

FIG. 17 a is a photocopy of a high-resolution TEM micrograph ofsilica-coated CdSe semiconductor nanocrystals in water.

FIG. 17 b shows the EDX mapping of silica-coated CdSe semiconductornanocrystals in water.

FIG. 18 a shows the photo-stability of CdSe semiconductor nanocrystalscoated with silica in PBS/H₂O with and without APS. For comparison, coresemiconductor nanocrystals in toluene are shown.

FIG. 18 b depicts the increase in percentage of Quantum Yield forsilica-coated CdSe Semiconductor nanocrystals relative to the uncoatedCdSe semiconductor nanocrystals in toluene after irradiation with 365 nmexcitation for 24 hours.

FIG. 19 graphically illustrates silica coating time vs. quantum yieldfor CdSe/ZnS semiconductor nanocrystals.

FIG. 20 is a photocopy of a micrograph illustrating single cores ofCdSe/ZnS encapsulated within single shells of silica having a diameterof about 25 nm.

FIG. 21 graphically illustrates coating time vs. quantum yield fordifferent water concentrations in the microemulsion.

FIG. 22 a is a photocopy of a micrograph showing consistent shellthickness, improved sphericity and improved monodispersity at increasingamounts of TEOS.

FIG. 22 b is a bar graph showing improved quantum yield with greateramounts of TEOS.

DETAILED DESCRIPTION

The present invention relates to nanoparticles, and methods of makingnanoparticles, that can exhibit improved quantum yield, and/or improvedwater solubility, and/or improved photo-stability, and/or improvedphoto-luminescence. The nanoparticles may include a non-semiconductorand/or non-metallic and/or non-inorganic passivation layer. Thepassivation layer may be amorphous, i.e., it lacks a crystallinestructure. One preferred form of amorphous passivation layer is onecomprising a material such as an amino silane. In addition, thenanoparticles may include a silica shell that partially or totallyencapsulates the nanoparticle core. In some cases, the silica shell maybe derivatized with, for example, polyethylene glycol (PEG) or othermaterials that provide improved characteristics, such as watersolubility. Nanoparticles of improved quantum yield, stability,solubility and/or biocompatibility can lead to improved methods, suchas, for example, medical diagnostics.

The term “nanoparticle” is used herein as it is known in the art andtypically refers to particles having a dimension of less of 100nanometers. One class of nanoparticles are the “semiconductornanocrystals” or “quantum dots” that can provide unique emission spectradependent, in part, on the size of the specific particle.

A “passivation” layer is a material associated with the surface of asemiconductor nanocrystal that serves to eliminate energy levels at thesurface of the crystal that may act as traps for electrons and holesthat degrade the luminescent properties of the nanocrystal. An exampleis a ZnS layer surrounding a CdSe semiconductor nanoparticle.Passivation layers result in improved quantum yields when compared tountreated particles.

An “emulsion” is a dispersion of a non-aqueous solvent and an aqueoussolvent. A “reverse emulsion” or “aqueous in non-aqueous emulsion” is adispersion of discrete areas of aqueous solvent (aqueous phase) within anon-aqueous solvent.

A “shell” is a layer that surrounds or partially surrounds ananoparticle core and in some cases may be chemically bound to thenanoparticle, such as by ionic or covalent bonding, and in other casesis not bound to the nanoparticle core. The shell forms part of thenanoparticle.

A “surfactant” is a material exhibiting amphiphilic properties and isused herein as it is commonly used in the art, e.g., for introducinghydrophobic species to hydrophilic environments.

The term “water soluble” is used herein as it is commonly used in theart to refer to the dispersion of a nanoparticle in an aqueousenvironment. “Water soluble” does not mean, for instance, that eachmaterial is dispersed at a molecular level. A nanoparticle can becomposed of several different materials and still be “water soluble” asan integral particle.

A “biological fluid” is a fluid present in or obtained from animal orplant and is typically aqueous in nature. Biological fluids include, forexample, blood, urine, lymph, saliva, sweat, and tears.

A “precursor” is a substance that can be transformed into a secondsubstance that exhibits different properties from the first. Forexample, a monomer is a polymer precursor if it can be transformed intoa polymer.

In one aspect, a nanoparticle includes a shell that encapsulates orpartially encapsulates the nanoparticle. In some embodiments, the shellis not chemically bound to the nanoparticle and yet may contain thenanoparticle by encapsulation. Thus, the nanoparticle and shell may bedevoid of ionic bonds and/or covalent bonds between the two. The shellmay be non-organic and may be a silicon polymer such as silica. Anon-organic shell is one that is not based on carbon and polymers ofcarbon, but nonetheless may, in some cases, include carbon atoms.

FIG. 1 shows schematically one embodiment of the invention. Nanoparticle100 includes a core 110 and a shell 120. Shell 120 can be chemicallybound or unbound to the core 110. The core can be a semiconductormaterial such as a semiconductor nanocrystal (quantum dot). The shellmay be generally spherical and may have a mean diameter that is about1.5×, 2×, 5×, 10× or >10× the diameter of the core. Typically, a singlecore is encapsulated by a single shell although in some embodiments, twoor more cores may be contained within a single shell.

In another aspect of the invention, a semiconductor nanocrystal includesa passivation layer. In some embodiments, the passivation layer may beof a material that is non-conductive and/or non-semiconductive. Forexample, the passivation layer may be of a material that does notexhibit a higher band gap than a nanocrystal which it surrounds. Inspecific embodiments, the passivation layer may be non-ionic andnon-metallic. A non-conductive material is a material that does nottransport electrons when an electric potential is applied across thematerial.

FIG. 3 illustrates schematically and not to scale a nanoparticle 200including a core 210, a surface passivation layer 230 and a shell 220.The shell may be organic or non-organic and may comprise a non-organicpolymer such as silica. In this embodiment, passivation layer 230 is anon-semiconductor and is preferably an amino (NH₂) silane, such asaminopropyl trimethoxysilane (APS). The inclusion of this passivationlayer has been shown to provide a quantum yield of about 10-30% or10-20% in aqueous media.

FIG. 4 illustrates schematically an amino silane modifying the surfaceof a nanocrystal core to form a passivation layer. The passivation layercan be comprised of, or consist essentially of, a compound exhibiting anitrogen-containing functional group, such as an amine. The amine may bebound directly or indirectly to one or more silicon atoms such as thosepresent in a silane or other silicon polymer. The silanes may includeany additional functional group such as, for example, alkyl groups,hydroxyl groups, sulfur-containing groups, or nitrogen-containinggroups. Compounds comprising the passivation layer may be of any sizebut typically have a molecular weight of less than about 500 or lessthan about 300. The preferred class of compounds are the amino silanesand in some embodiments, amino propyl trimethoxysilane (APS) can beused. The use of APS in semiconductor nanocrystals has been shown toprovide passivation and to improve quantum yields to a level comparableto the improvements obtained by the use of higher band gap passivationlayers such as those made of zinc sulfide (ZnS).

Nanoparticles including a silica shell will, of course, be of greaterdimensions than a similar nanoparticle absent the shell. For example,nanoparticles of the invention may exhibit mean diameters of less than100 nm, less than 50 nm, or less than or equal to about 25 nm. In otherembodiments, the mean diameter of the nanoparticle including the shellmay be greater than 5 nm, greater than 10 nm, greater than 20 nm, orgreater than or equal to about 25 nm.

In some embodiments, a silica shell can be functionalized or derivatizedto include compounds, atoms, or materials that can alter or improveproperties such as water solubility, water stability, photo-stabilityand biocompatibility. For example, a silica shell can include moietiessuch as polyethylene glycol (PEG) and other glycols. Thesenanoparticles, with and without PEG, have been shown to be non-toxic toliving cells for extended periods, and it is believed that thenanoparticles are also non-toxic in vivo due, at least in part, to theisolation of the toxic core within the polymerized silica shell.

As shown in FIG. 2, a shell 120, that may be non-organic, can includehydrophilic species 130 that can provide greater hydrophilicity to thenanoparticle. Hydrophilic species 130 can be, for example, apolyethylene glycol (PEG) or a derivative of polyethylene glycol.Derivatives include, but are not limited to, functionalized PEGs, suchas amine, thiol and carboxyl functionalized PEG. The hydrophilic species130 is associated with the shell 120 and can be chemically bound to theshell 120 or can be, for example, physically trapped by the shellmaterial. Preferably, hydrophilic species 130 includes a portion thatcan be chemically bonded to the shell and a second portion that provideshydrophilicity and may extend outwardly from the surface of the shell.

Presence of these glycols can impart superior water solubilitycharacteristics to the nanoparticles while being biocompatible andnontoxic and can, in some instances, provide for better dispersion ofthe nanoparticles in solution. For example, by integrating PEG into thesilica shell, the semiconductor nanocrystal may be rendered watersoluble at pHs of less than 8, less than or equal to 7.5, less than orequal to 7 or less than or equal to 6.5. Thus, these nanoparticles maybe water soluble at neutral or below neutral pHs and thus may bebiocompatible and appropriate for use in biological fluids such as bloodand in vivo. In some embodiments, the inclusion of PEG into the silicashell provides for a nanoparticle that can remain in solution for aperiod of greater than 1 hour, greater than 6 hours, greater than 12hours or greater than 1 day. In addition, the presence of PEG or relatedcompounds in the silica shell can provide for a nanoparticle exhibitinga reduced propensity to adsorb protein, cells, and other biologicalmaterials. This means that, for example, when used in vivo, theparticles can stay in solution for a longer period of time than dosimilar particles, thus allowing for increased circulation and improveddeliverability to intended targets.

One embodiment is shown in FIG. 5 that illustrates schematically theformation of a silica shell 120 around a CdSe core 110. Portions of twosurfactants, TOPO 140 and IGEPAL 150, are shown. TOPO includes thehydrophilic group phosphine oxide and IGEPAL includes the hydrophilicgroup PEO. The source of the IGEPAL 150 is the reverse microemulsion(aqueous in non-aqueous) and the source of the TOPO is the TOPO cappedsemiconductor nanocrystal. FIGS. 6 a-6 c illustrate how the reversemicelles can present the nanocrystal cores for encapsulation at thewater/oil interface. Thus, while the nanocrystals may be transported tothe aqueous phase, the formation of the silica polymer tends to occuraround the nanocrystals at the aqueous/non-aqueous interface. FIGS. 7a-7 c illustrate the formation of a silica shell around a nanoparticlecore at the oil/water (cyclohexane/water) interface.

The aqueous in non-aqueous microemulsion can be produced using a varietyof non-polar solvents. Preferably the non-polar solvent is a hydrocarbonand may be an aliphatic hydrocarbon and in a more preferred embodimentis a non-aromatic cyclic hydrocarbon such as cyclopentane, cyclohexaneor cycloheptane.

In one embodiment, a semiconductor nanocrystal including a core of acadmium chalcogenide is coated with a passivation layer comprising anamine. The core, that may include cadmium selenide or cadmium telluride,for example, can be made using methods known to those skilled in theart. An aqueous in non-aqueous reverse micro emulsion can be prepared,using for example, an ionic or non-ionic surfactant. Non-ionicsurfactants include, for example, polyphenyl ethers, such as IGEPALCO-520, while ionic surfactants include, for example, dioctylsulfosuccinate sodium salt (AOT). As conventionally prepared, thecalcium chalcogenide core is typically presented with a trioctylphosphine oxide (TOPO) surfactant. TOPO includes a hydrophilic endcomprising phosphine oxide while IGEPAL includes a hydrophilic endcomprising polyoxyethylene (PEO). After introduction of the TOPOsemiconductor nanocrystals into the reverse emulsion, the TOPO can bepartially or completely exchanged for IGEPAL due, in part, to the muchhigher concentration of IGEPAL in the reverse emulsion.

Upon the exchange of TOPO for IGEPAL, the semiconductor nanocrystalcores are amenable to the aqueous domains in the reverse emulsion and asol-gel precursor, such as tetraethylorthosilicate (TEOS) can bepolymerized using methods known to those skilled in the art, around thecore to produce a silica shell. The resulting nanostructure includes acore of a cadmium chalcogenide, a passivation layer of an amino silanesuch as APS and a hydrophilic shell, such as a shell of polymerizedsilica. Surfactants other than IGEPAL may be used and may be varied, inpart, depending upon the core material, how the nanoparticle core iscapped and the reverse emulsion that is used. Preferred surfactants arethose that can be exchanged for TOPO or other surfactants that are usedto cap the core and that also provide enough hydrophilicity to draw thecore into aqueous portions of the micro-emulsion, thus providing anenvironment for the formation of the silica shell.

In another aspect, a method of making a nanoparticle including a silicashell modified to improve bio compatibility and/or water solubility isprovided. For example, in some embodiments a PEG modified silica shellcan be formed around a nanoparticle. The nanoparticle core may be asemiconductor nanocrystal or other nanoparticle. As described above, thenanoparticle core may be introduced into a reverse micro-emulsion(aqueous in non-aqueous emulsion) to prepare it for encapsulation. Inanother step, a base such as ammonia (NH₄OH) including a glycol such aspolyethyleneglycol monomethylether (PEG-m) can be dissolved into themicroemulsion. The PEG may be of any molecular weight, but it ispreferably of a molecular weight of greater than 1,000 and less than20,000 and in some embodiments, is in a range of between 5,000 and10,000. A sol-gel precursor such as TEOS can then be added and themixture can be stirred allowing the PEG to be incorporated into theforming silica shell. The resulting silica shell derivatized with PEGcan provide for improved quantum yield, improved water solubility,improved biocompatibility in a reduced propensity to coagulate.

In one embodiment, ammonia and PEG are stirred into the microemulsionbefore a sol-gel precursor such as TEOS is added. After addition of thesol-gel precursor, the microemulsion can be stirred continuously until apreferred amount of silica polymerization has taken place. During thistime, the PEG is incorporated into the silica shell and can alter theproperties of the silica shell by, for example, increasinghydrophilicity, altering the nanoparticle's propensity to adsorbmaterials such as proteins in cells, and can increase repulsion forcesbetween particles, providing for an extended period of suspension of theparticles without coalescing.

The amount of water (29.5% aqueous NH₄OH) in the aqueous in non-aqueous(reverse) microemulsion can be varied based upon the specific reactionthat is desired. For example, in some embodiments the amount of water inthe reverse microemulsion is between 0.2 and 0.5 percent by volume. Inpreferred embodiments, the amount of water is between 0.3 and 0.4percent by volume and in some embodiments it has been found that quantumyield can be maximized when the amount of water in the reversemicroemulsion is about 0.3 percent by volume.

The amount of sol-gel precursor added to the microemulsion can alsoaffect the properties of the nanoparticle. For example, while anincrease in the amount of sol-gel precursor does not appear to increasethe shell thickness, an increase in the amount of sol-gel precursor doesappear to improve this sphericity as well as the monodispersity of theparticles. In some embodiments, quantum yield is also improved withhigher concentrations of sol-gel precursor. For example, see FIGS. 20Aand 20B.

EXAMPLES Example 1 Preparation of Silica-Coated ZnS-Capped CdSeNanocrystals (CdSe/ZnS/SiO₂) in Reverse Microemulsion Aqueous inNon-Aqueous Emulsion

CdSe/ZnS semiconductor nanocrystals with excess TOPO without any surfacemodification were prepared according to literature procedures (Hines etal., “Synthesis and Characterization of Strongly Luminescing ZnS-CappedCdSe Nanocrystals” J. Phys. Chem. 100, 468-471, 1996. Dabbousi et al.,“(CdSe)ZnS Core-Shell Quantum Dots: Synthesis and Characterization of aSize Series of Highly Luminescent Nanocrystallites” J. Phys. Chem. B101, 9463-9475, 1997.). The particles had luminescence quantum yields of10-25% and emission at 625 nm (FWHM=30 nm). The particles wereprecipitated once from methanol to remove excess TOPO andtrioctylphosphine oxide (TOP). Inverse micelles were prepared using anon-ionic surfactant such as Igepal CO-520 and cyclohexane as solvent.

FIGS. 6 and 7 depict the schemes of the transformation of hydrophobicsemiconductor nanocrystal into aqueous domains of the reversemicroemulsion and silica coating, respectively. Typically, core-shellsemiconductor nanocrystals passivated with TOPO and dissolved either inbutanol or n-hexane were injected into the reverse micelles. This wasfollowed by the addition of TEOS and was allowed to stir for 1 hour.Addition of ammonia resulted in a stable water-in-oil reversemicroemulsion. The resulting solution was stirred for 24 hours, whichresulted in homogeneous silica deposition. Due to the large excess ofIgepal, the TOPO ligand was exchanged for Igepal in cyclohexane and thesemiconductor nanocrystals became more hydrophilic. The nanocrystalswere then solubilized by water through exchange of the Igepal-cappedsemiconductor nanocrystals with Igepal-capped aqueous domains.

Example 2 TEM Characterization of Silica-Coated ZnS-Capped CdSeNanocrystals (CdSe/ZnS/SiO₂) in Reverse Microemulsion

Electron microscopy revealed that the majority (>90%) of nanocrystalsare encapsulated as single particles within a silica shell (FIG. 8). Theaverage total diameter of nanocrystal/Silica is ˜22 nm. FIG. 9 shows theencapsulation of nanocrystals within ca. 100 nm silica in the reversemicroemulsion. Varying the amount of water and ammonia in themicroemulsion also affects the shell thickness, since this alters theaqueous domain size. The silica shell thickness can be increased byincreasing the TEOS concentration as shown in FIG. 10. The higher theconcentration of TEOS the larger is the silica diameter. Increase in thesize of silica lowers the emission intensity and QY. The total diameterof coated nanocrystals affect the QY relative to the uncoatednanocrystals, which is also shown in FIG. 10.

Example 3 Emission Characteristics of Silica-Coated ZnS-Capped CdSeNanocrystals (CdSe/ZnS/SiO₂) in Water

Silica-coated nanocrystals in the microemulsion were centrifuged at18000 rpm for 30 minutes and the pellet was washed with cyclohexanetwice and dispersed in an alkaline aqueous solution of pH 8-9. Thesilica-coated nanocrystals in water were characterized by UV-visibleabsorption spectroscopy and fluorescence spectroscopy. FIG. 11 shows theabsorption and fluorescence spectra of the CdSe nanocrystals before andafter silica coating. UV-visible and photoluminescence spectra show thatthere is a 5 nm red-shift for CdSe/ZnS/SiO₂ in water, in comparison tothe CdSe/ZnS nanocrystals in the parent solution, butanol. Silica-coatedsemiconductor nanocrystals in water showed remarkable colloidalstability over a period of several months.

Silica-coated nanocrystals showed remarkable photostability. Thenanocrystals in water and in butanol were exposed to UV irradiation (335nm cut-off filter). About 85% of the quantum yield was retained after 24hours for the coated particles in water, where as the parentsemiconductor nanocrystal solution virtually lost all luminescence after24 hours. We also compared the results with those dots in water cappedby mercaptoundecanoic acid (MUA). MUA-capped semiconductor nanocrystalsin water exhibited initial photo-brightening and then photo-dissolutionover a period of 24 hours. The exciton absorption peak position isplotted against time of photolysis in FIG. 12. The peak position remainsconstant at 616 nm for silica-coated semiconductor nanocrystals,indicating that the dots are photostable over extended periods ofphotolysis time. On the contrary, the nanocrystals capped with MUA showphoto-dissolution as exemplified by blue-shifts in the exciton positionover 24 hours of photolysis.

Example 4 Preparation of TOPO-Capped CdSe Semiconductor Nanocrystals andInteraction of Surfactants in IGEPAL Reverse Micelles

The following experiment was run to demonstrate that the TOPO on a TOPOcapped nanocrystal could be exchanged for a more hydrophilic surfactantthat can alter the hydrophilicity of the nanoparticle.

CdSe semiconductor nanocrystals with excess TOPO without any surfacemodification were prepared according to literature procedures (Peng etal., “Formation of High-Quality CdTe, CdSe, and CdS Nanocrystals UsingCdO as Precursor” J. Am. Chem. Soc. 123, 183-184, 2001). The particleshad luminescence quantum yields of 10-20% and emissions at 550-600 nm(FWHM=30-40 nm). The particles were precipitated once from methanol toremove excess TOPO and trioctylphosphine oxide (TOP). Inverse micelleswere prepared using the non-ionic surfactant IGEPAL CO-520(polyoxyethylene nonylphenyl ether), at 5%, by weight, and cyclohexaneas solvent.

Emission experiments showed that TOPO-capped nanocrystals were exchangedby IGEPAL. Changes in emission intensity and emission wavelength fornanocrystals in IGEPAL micelles in comparison with those nanocrystals incyclohexane are due to the surface ligand exchange reactions betweenTOPO and IGEPAL.

Example 5 Preparation of Silica-Coated Nanocrystals (CdSe/SiO₂) inIGEPAL Reverse Microemulsion Aqueous in Non-Aqueous Emulsion

CdSe nanocrystals passivated with TOPO were precipitated with methanolonce and the precipitate was dried under nitrogen in order to remove themethanol. To the precipitate, cyclohexane was added and vortexed untilthe solution became clear. The semiconductor nanocrystals wereintroduced into the reverse micelles of an aqueous in non-aqueousemulsion made with 0.5 g IGEPAL and 10 ml cyclohexane, and stirred for30 minutes. Then 100 μl of 29.5% NH₄OH was added and stirred for another1 hour. Finally, TEOS was added at different concentrations and thestirring continued for 24 hours. Aliquots of samples were taken atdifferent periods of coating times ranging from 1 to 24 hours, andemission spectra were recorded.

FIGS. 13 a-13 c depict the effect of TEOS concentration and silicacoating time on the emission characteristics. For comparison, theemission spectra of nanocrystals in IGEPAL micelles (no silica coating)are shown. It is clear that the full width at half maximum (FWHM) of theemission peak is increased with an increase in coating time. In FIG. 14a the relative emission intensity is plotted against coating time atvarious TEOS concentration. Results show that the emission intensityincreases at 2 hours of silica coating after an initial decrease at 1hour, and that a further increase in coating time at 4 hours decreasesthe emission intensity, which remains saturated with further increase incoating time. The initial increase in the intensity at 2 hours isapparently due to silica formation when ammonia adsorption occurs on thesurface sites of the nanocrystals. The decrease in emission intensity ispresumably due to the acidity of the silanol groups that quench ammoniabasicity by a desorption process at higher coating times (FIG. 14 a).

FIG. 14 b shows the effect of coating time on the emission wavelength atdifferent TEOS concentrations and different particles sizes. In all thecases, silica-coated nanocrystals exhibit a blue shift in comparisonwith naked (uncoated) dots. This implies that IGEPAL-capped nanocrystalsare exchanged for silica-coated nanocrystals. The surface exchangereaction accounts for the blue shift. The best conditions for silicacoating are found to employ 5 μl TEOS in a total volume of 10 mlmicroemulsion and 2 hours of coating time (FIG. 14 c).

Example 6 Preparation of Silica-Coated Nanocrystals (CdSe/SiO₂) inIGEPAL Reverse Microemulsion with an Amino Silane, APS, Added

CdSe semiconductor nanocrystals passivated with TOPO were precipitatedwith methanol once and the precipitate was dried under nitrogen in orderto be free from methanol. 2 μL of APS were added to the precipitate,which was dissolved in 1 ml of cyclohexane and vortexed until thesolution became clear. APS-modified nanocrystals were introduced intothe reverse micelles and stirred for 30 minutes. 100 μl of 29.5% NH₄OHwas the added and stirred for another 1 hour. Finally, 5-20 μl TEOS wasadded and the stirring continued for 24 hours. Aliquots of samples weretaken at different periods of coating times ranging from 1 to 24 hoursand emission spectra were recorded for samples from each aliquot (FIGS.15 a-15 c).

The results show that APS modification provides more surface passivationthan is found in the naked dots. The APS modification is preferably donebefore injecting the nanocrystals into IGEPAL reverse micelles. Withsilica coating, there is no initial decrease in the emission intensityat lower TEOS concentrations (FIGS. 15 a and 15b), but there is adecrease at higher concentrations (FIG. 15 c). At 8 hours of coatingtime, the emission intensity remains higher than all other coating timesup to 24 h. The comparison of the emission intensities at 8 and 24 hoursshows a negligible decrease (FIG. 16 a) irrespective of TEOSconcentration. This is believed to occur because the amine groupsprovide additional surface passivation for the APS-modified nanocrystalsin comparison to the similar nanocrystals absent the APS modification.Furthermore, the acidity of the silanol groups that are formed duringextended periods of silica coating does not seem to affect the emissionproperties as the amine protects the nanocrystal surface.

FIG. 16 b illustrates the effect of coating time on the emissionwavelength for different TEOS concentrations and different particlessizes of APS-modified nanocrystals. Although silica-coated nanocrystalsexhibit a blue shift (5-10 nm) in comparison with naked (uncoated)nanocrystals at different coating times and various TEOS concentrations,the observed blue shifts are small compared to those for unmodifiednanocrystals (10-20 nm) as shown in FIG. 13 b. From this data, goodconditions for silica coating are found to be 2 μl TEOS and 8 hours ofcoating time (FIG. 16 c).

Example 7 TEM Characterization of Silica-Coated Nanocrystals (CdSe/SiO₂)in Water

The silica-coated colloidal solution was centrifuged at 12000 rpm for 20minutes and the resulting pellet was washed with deionized water twiceand dispersed in a mixture of water and phosphate buffered saline (PBS).FIG. 17 a shows the HRTEM image of silica-coated nanocrystals in water.The lattice fringes of CdSe are marked by arrows. The energy dispersiveanalysis of X-rays (EDX) mapping (FIG. 17 b) confirms the presence ofall elements that are predicted: Cd, Se from CdSe semiconductornanocrystal core, Si, O from SiO₂ shell and P from TOPO. Sulfurimpurity, possibly from surfactant, is also seen.

Example 8 Photo-Stability of Silica-Coated Nanocrystals (CdSe/SiO₂) inWater

FIG. 18 a shows the photo-stability of three different semiconductornanocrystals. Included are silica-coated nanocrystals without APS andsilica-coated nanocrystals with APS, both in a mixture of water andphosphate buffered saline (PBS). The third nanocrystal is an uncoatednanocrystal (no silica) in toluene. As can be seen, the coated dotsexhibited superior photo-stability than did the uncoated ones. Theincrease in QY relative to the uncoated nanocrystals in toluene isbelieved to be due to the photo-brightening of silica-coatednanocrystals. This may be caused by the photo-ionization of thenanocrystals. An approximately three-fold increase in QY forsilica-coated nanocrystals with APS modification before coating,compared to silica-coated nanocrystals without APS modification, isclearly seen in FIG. 18 b.

Example 9 Preparation of PEG-Silica-Coated Nanocrystals (CdSe/ZnS/SiO₂and CdSe/SiO₂) in IGEPAL Reverse Microemulsion with No APS Added

CdSe/ZnS and CdSe nanocrystals passivated with TOPO were precipitatedwith methanol once and the precipitate was dried under nitrogen in orderto be free from methanol. To the precipitate, cyclohexane was added andvortexed until the solution became clear. The nanocrystals wereintroduced into an aqueous in non-aqueous emulsion, as described above,and stirred for 30 minutes. 50 μl NH₄OH containing polyethylene glycolmonomethyl ether (PEG-m) concentration of 0.05 g/ml NH₄OH, was added andstirred for another 1 hour. Finally, TEOS was added at different amountsand the stirring continued for 144 hours. Aliquots of samples were takenat different periods of coating times ranging from 24 to 144 hours. Thesamples were then transferred to water, and the resulting suspensionsremained stable, even after 3 weeks. The enhanced solubility isattributed to the repulsion force and solvation layer provided by thePEG. The quantum yield associated with different coating times was alsorecorded.

FIG. 19 illustrates the effect of silica coating time of CdSe/ZnSnanocrystals on the quantum yield of the samples. The quantum yield isplotted against coating time. The results show that the emissionintensity increases to a maximum after 90 hours of silica coating. Themaximum quantum yield (17%) achieved is greater than that of thepre-coated CdSe/ZnS (10%). This is attributed to enhanced surfacepassivation by the silica coating layer surface moiety.

FIG. 20 provides an electron micrograph showing that the CdSe/ZnS isencapsulated as single particle within a silica shell of diameter ˜25nm. Varying the amount of water (0.3% to 0.4%) in the microemulsion doesnot appear to affect the shell thickness but does appear to decrease thequantum yield (FIG. 21). Varying the volume of TEOS added (20-60 μl)does not appear to affect the shell thickness but does seem to improvethe sphericity and monodispersity of the particles, as well as thequantum yield, as evidenced by FIGS. 22 a and 22b.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified. Thus, as a non-limiting example, a reference to“A and/or B” can refer, in one embodiment, to A only (optionallyincluding elements other than B); in another embodiment, to B only(optionally including elements other than A); in yet another embodiment,to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, unless clearlyindicated to the contrary, “or” should be understood to have the samemeaning as “and/or” as defined above. For example, when separating itemsin a list, “or” and “and/or” each shall be interpreted as beinginclusive, i.e., the inclusion of at least one, but also including morethan one, of a number or list of elements, and, optionally, additionalunlisted items. In general, the term “or” as used herein shall only beinterpreted as indicating exclusive alternatives (i.e. “one or the otherbut not both”) when preceded by terms of exclusivity, such as “only oneof” or “exactly one of.”

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements that the phrase “at least one” refers to, whether related orunrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

1. A semiconductor nanocrystal solution comprising: an aqueous solutionhaving a pH of less than about 8.0; a plurality of semiconductornanocrystals dissolved in the aqueous solution wherein at least 90% ofthe semiconductor nanocrystals, by weight, remain dissolved for greaterthan 6 hours.
 2. The semiconductor nanocrystal solution of claim 1wherein the aqueous solution is a biological fluid.
 3. The semiconductornanocrystal solution of claim 1 wherein the aqueous solution ismammalian blood.
 4. The semiconductor nanocrystal solution of claim 1wherein the aqueous solution has a pH of less than equal to 7.0.
 5. Thesemiconductor nanocrystal solution of claim 1 wherein the semiconductornanocrystals comprise polyethylene glycol.
 6. A semiconductornanocrystal wherein the nanocrystal is soluble in water at a pH of lessthan about 8.0.
 7. The semiconductor nanocrystal of claim 6 wherein thenanocrystal comprises a shell comprising silica.
 8. The semiconductornanocrystal of claim 7 wherein the shell includes a polyethylene glycol.9. The semiconductor nanocrystal of claim 8 wherein the polyethyleneglycol has a molecular weight of about 5,000 to 10,000.
 10. Ananoparticle comprising a silica shell encapsulating a core, the silicashell including polyethylene glycol, or a derivative thereof.
 11. Thenanoparticle of claim 10 wherein the polyethylene glycol has a molecularweight of about 5,000 to about 10,000.